A bacillus type composite microbial agent, a preparation method and application thereof

Abstract

The application discloses a bacillus type composite microbial agent and a preparation method and application thereof, and relates to the field of agricultural biotechnology; comprising the following steps: S1: strain selection and culture; S11: strain screening, screening out the baciilus subtilis Z-14 and the bacillus mucilaginosus HB-02 which have vigorous growth vigor and resistance to adversity and heat, inoculating the screened strains on NA culture medium respectively, culturing overnight in constant temperature, picking activated single colonies and transferring into NB culture medium for oscillation culture overnight to prepare bacterial suspension; S12: bacterial activation, carrying out bacterial activation by using NA culture medium and culturing overnight in constant temperature; the composite microbial agent can effectively prevent and treat the occurrence of wheat root rot, and the effect is better than that of using the Z-14 agent and the HB-02 agent alone; field experiments show that the prevention and treatment effect of the composite microbial agent on the wheat root rot reaches 81.8%, and is significantly better than that of the single microbial agent.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a Bacillus-type compound microbial agent and its preparation method. Background Technology

[0002] Compound microbial agents refer to agents that include one or more beneficial microorganisms and microbial carriers, and contain a certain number of live bacteria. They can quickly replenish beneficial bacteria in the soil, inhibit harmful bacteria, regulate soil pH, improve soil fertility, enhance plant resistance to diseases and pests, and promote plant growth. They can effectively overcome the shortcomings of using single microbial agents.

[0003] For example, Li Xueping et al. reported a compound bacterial agent BP5 composed of cold-resistant short bacillus (Brevibacterium frigoritolerans), Bacillus cereus (B. cereus), and Bacillus subtilis. After field application, it achieved a control efficacy of 68% against lily wilt disease and effectively improved lily quality and soil quality.

[0004] However, these compound microbial agents are all formulated in equal proportions, and their optimal ratio is still unclear, limiting the synergistic effect of the strains. Furthermore, the field application effects and optimal dosage are not well understood, hindering their large-scale demonstration and widespread application. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a Bacillus-type composite microbial agent and its preparation method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A type of Bacillus, specifically a complex Bacillus, comprising:

[0008] Bacillus subtilis, accession number CGMCC No. 20821; depository center: China General Microbiological Culture Collection Center; address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: September 25, 2020;

[0009] Paenibacillus mucilaginosus, accession number CGMCC No. 24636; depository center: China General Microbiological Culture Collection Center; deposit address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: April 6, 2022.

[0010] A method for preparing a Bacillus-type compound microbial agent includes the following steps:

[0011] S1: Strain selection and culture;

[0012] S11: Bacillus subtilis Z-14 and Bacillus mucilaginosus HB-02, which have strong growth vitality and are resistant to stress and heat, were screened out. The selected strains were streaked on NA medium and incubated overnight at constant temperature. Activated single colonies were picked and transferred to NB medium and shaken overnight to prepare bacterial suspension.

[0013] S12: Bacterial activation, using NA medium for bacterial activation, and incubating overnight at a constant temperature;

[0014] S13: Cell collection and preparation: Activated single colonies were inoculated into NB medium and cultured overnight with shaking. The culture was then inoculated into fresh NB medium at a 10% inoculation rate and cultured with shaking for 48 hours. The fermentation broth was centrifuged, the cells were recovered, and diluted with sterile water.

[0015] S2: Preparation of compound microbial agents;

[0016] S21: Determine the mixing ratio, conduct tests, and determine the optimal mixing ratio of Z-14 and HB-02 bacterial agents based on the test results;

[0017] S22: Prepare the bacterial suspension by adjusting the viable bacteria content of the Z-14 and HB-02 bacterial agents, which are mixed in proportion, to 1.0 × 10⁻⁶. 10 A compound bacterial suspension was prepared by adding cfu / mL.

[0018] Preferably, in step S11, the viable bacteria content of the bacterial suspension is controlled to be 1×10⁻⁶. 8 cfu / mL or 1×10 10 cfu / mL.

[0019] Preferably, the specific components of the NA culture medium are: 5g beef extract, 10g peptone, 5g NaCl, 15-20g agar, and 1000mL water;

[0020] The pH of the NA medium should be controlled between 7.0 and 7.2.

[0021] Preferably, the specific components of the NB culture medium are: 5g beef extract, 10g peptone, 5g NaCl, and 1000mL water;

[0022] The pH of NB medium should be controlled between 7.0 and 7.2.

[0023] Preferably, in step S13, the centrifugation speed is controlled at 8000 rpm and the centrifugation time is controlled at 10 minutes.

[0024] Preferably, in step S13, the bacteria are diluted with sterile water to a viable bacterial count of 1×10⁻⁶. 8 cfu / mL or 1×10 10 cfu / mL.

[0025] Preferably, in step S21, the specific method for conducting the test includes:

[0026] Pot experiment: A pot experiment was conducted by setting up a blank control group, a pathogen group, and different treatment groups to test the control effect and growth-promoting effect of each treatment on wheat root rot.

[0027] Field trials: Field trials were conducted under actual farmland conditions to determine the effects of compound microbial agents on wheat yield, plant height, number of ears, number of grains per ear, and soil enzyme activity;

[0028] Effect analysis and ratio optimization: Combining the results of pot and field trials, the effects of different proportions of compound microbial agents were analyzed, and the optimal mixing ratio was finally determined.

[0029] Preferably, in S21, the optimal mixing ratio is Z-14 and HB-02 bacterial agents mixed at a ratio of 2:1.

[0030] Preferably, in step S1, the temperature for overnight constant temperature incubation and shaking incubation is controlled at 30°C.

[0031] Preferably, in the pathogen group, the pathogen culture medium is PDA medium, which specifically consists of: 200g potato, 20g glucose, 15-20g agar, and 1000mL water. Peel the potatoes, cut them into small pieces, boil them in water for 0.5 hours, filter through double-layered gauze, add sugar to the filtrate, and add water to make up to 1000mL.

[0032] Melt the sterilized PDA medium and pour it into plates. Add streptomycin sulfate to bring the final concentration to 40 μg / mL. Inoculate the pathogenic bacteria onto the center of the PDA plate and incubate at 26°C until the mycelium has fully grown on the plate.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. The compound microbial agent of the present invention can effectively prevent and control the occurrence of wheat root rot, and its effect is better than that of using Z-14 agent and HB-02 agent alone; field experiments show that the compound agent treatment group has a control effect of 81.8% on wheat root rot, which is significantly better than the effect of single agent.

[0035] 2. Inoculation with Z-14 and HB-02 microbial agents can significantly increase wheat yield and promote growth. The compound microbial agent treatment group showed the best yield increase in all test indicators, with a yield increase of 18.19% compared to the control group.

[0036] 3. The compound microbial agent of this invention can improve soil fertility, regulate soil physicochemical properties, increase soil enzyme activity, and improve the cultivation effect of wheat; by supplementing beneficial bacteria in the soil, inhibiting harmful bacteria, and improving the soil microenvironment, it can improve soil quality.

[0037] 4. The biological control method of this invention is green and environmentally friendly, and pathogens are less likely to develop drug resistance. Compared with chemical fungicides, the use of compound microbial agents will not cause environmental pollution or pesticide residues in agricultural products, which meets the requirements of green agricultural development. Attached Figure Description

[0038] Figure 1 This is a flowchart of a method for preparing a Bacillus-type composite microbial agent proposed in this invention. Detailed Implementation

[0039] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0040] Example 1:

[0041] A Bacillus-type compound microbial agent, the preparation, testing, and application of which specifically include:

[0042] 1. Materials and Methods

[0043] 1.1 Preparation of experimental materials

[0044] Soil samples were taken from wheat-growing areas at the Hebei Agricultural University farm. The wheat variety used in the experiment was Jimai 418, which originated in Baoding and was uncoated.

[0045] 1.2 Culture medium

[0046] PDA medium: 200g potato, 20g glucose, 15-20g agar, 1000mL water. Peel the potato, cut it into small pieces, boil it in water for 0.5h, filter it through double-layer gauze, add sugar to the filtrate, and add water to make up to 1000mL.

[0047] NA medium: 5g beef extract, 10g peptone, 5g NaCl, 15-20g agar, 1000mL water, pH 7.0-7.2.

[0048] NB medium: 5g beef extract, 10g peptone, 5g NaCl, 1000mL water, pH 7.0-7.2.

[0049] 1.3 Pathogen Culture

[0050] The wheat root rot pathogen *Cochliobolus sativus* was preserved by the Agricultural Microbiology Laboratory of the College of Life Sciences, Hebei Agricultural University. Sterilized PDA medium was melted and poured into plates, with streptomycin sulfate added to a final concentration of 40 μg / mL. The pathogen was inoculated onto the center of a PDA plate and incubated at 26°C until the mycelium had fully colonized the plate.

[0051] 1.4 Bacterial Culture

[0052] A small amount of bacterial colony was picked from the NA slant using a sterile bamboo stick and streaked onto a fresh NA agar plate. The plate was incubated overnight at 30°C. Activated single colonies were then inoculated onto NB agar and incubated overnight with shaking at 30°C. Finally, the culture was inoculated at a 10% inoculum onto fresh NB agar and incubated with shaking at 30°C for 48 hours. The fermentation broth was centrifuged at 8000 rpm for 10 minutes. The bacterial cells were recovered and diluted with sterile water to obtain a viable cell count of approximately 1 × 10⁻⁶. 8 cfu / mL or 1×10 10 The viable cell count was determined using a bacterial suspension containing cfu / mL and a bacterial counting chamber.

[0053] 1.5 The effect of microbial inoculants on the control of wheat root rot

[0054] Five treatments were set up for the pot experiment: a blank control group (CK0), a pathogen group (CK1), a Z-14 inoculant treatment group (Z14), an HB-02 inoculant treatment group (HB02), and a Z-14 and HB-02 compound inoculant treatment group (ZHB, a 1:1 mixture of Z-14 and HB-02 inoculants). Each treatment had five replicates. Pots with a diameter of 12.9 cm and a height of 12.4 cm were used, and 12 wheat seeds were evenly sown in each pot. CK0 was not inoculated with the pathogen or antagonistic bacteria. CK1 was inoculated with wheat root rot pathogen, with wheat seeds placed on pathogen substrate sheets (7 mm in diameter), one wheat seed per substrate. The Z14, HB02, and ZHB treatment groups were inoculated with the pathogen in the same way as CK1, and then 20 mL of Z-14, HB-02, and the mixed bacterial suspension were applied, respectively. The plants were incubated at room temperature, and the control effect of the antagonistic bacteria was investigated after 20 days of cultivation. Plant growth status was assessed on a per-plant basis, with a sample size of 20 plants, and the average value was calculated. The severity grading standard for wheat root rot was as follows: Grade 0: No disease; Grade 1: The outer leaf sheath at the base of the wheat stem turns blackish-brown, and the lesion length is less than 1 / 2 of the leaf sheath; Grade 2: The outer leaf sheath at the base of the wheat stem turns blackish-brown, and the lesion length is greater than 1 / 2 of the leaf sheath; Grade 3: The inner leaf sheath at the base of the wheat stem turns blackish-brown, and the lesion length is less than 1 / 2 of the leaf sheath; Grade 4: The inner leaf sheath at the base of the wheat stem turns blackish-brown, and the lesion length is greater than 1 / 2 of the leaf sheath; Grade 5: Root rot, plant death. Disease index = [∑(Number of diseased plants at each level × Representative value) / Total number of plants surveyed × Representative value of the most severe disease level] × 100. Control effect = [(Disease index of control group - Disease index of treatment group) / Disease index of control group] × 100.

[0055] 1.6 Field Trial of the Growth-Promoting Effect of Microbial Inoculants

[0056] A wheat planting plot was selected at the experimental farm of Hebei Agricultural University, with an area of ​​approximately 800 square meters. 2 It is divided into 4 small pieces, each piece being approximately 200m². 2 The groups were designated as follows: blank control group CK0, Z-14 inoculant treatment group Z14, HB-02 inoculant treatment group HB02, and Z-14 and HB-02 compound inoculant treatment group ZHB (a 1:1 mixture of Z-14 and HB-02 inoculants). The control group received no inoculant; the other three groups received inoculants with a viable bacterial count of approximately 1.0 × 10⁻⁶. 10 Treat seeds with a CFU / mL inoculum solution at a rate of 20 mL / kg of seeds; during the greening stage, inoculate again with the inoculum solution by irrigation at a concentration of 1.0 × 10⁻⁶. 8 CFU / mL, inoculation dose is 1000 mL / m 2Neither the control nor the treatment group received fertilizer. Mechanical sowing was used, and management methods were the same as in general field operations. During the milk stage of wheat, a 5-point sampling method was used, with 3 consecutive rows sampled at each point, and each row sampled at 0.4m. Plant height and average number of grains per ear were investigated and recorded (30 plants at each random sampling point, 150 plants per group). After the seeds were dried, the thousand-grain weight was measured, and the yield per square meter was determined by individual harvesting.

[0057] 1.7 Optimization of Compound Microbial Agent Ratio

[0058] Z-14 and HB-02 bacterial agents were mixed in different ratios. The treatment group with a live bacteria content ratio of 3:1 for Z-14 and HB-02 was designated as TR1, TR2 as TR2, TR3 as TR3, TR4 as TR4, and TR5 as TR5. The blank control group without bacterial agents was designated as CK0, and the pathogen group with pathogens was designated as CK1. The disease prevention and growth promotion effects of different ratios of compound bacterial agents were tested. The disease prevention effect was tested using the same method as in 1.5, and the growth promotion effect was tested using the same method as in 1.6.

[0059] 1.8 Soil Enzyme Activity Determination

[0060] In a field trial investigating the effects of compound microbial agents on wheat growth, the activities of bioenzymes in the rhizosphere soil of wheat from different treatment groups were measured at the wheat jointing stage. Sucrase activity was determined using a colorimetric method, with sucrase activity expressed as the mass (mg) of glucose produced in 1.0g of soil after 1 day, expressed in mg·g⁻¹. -1 ·d -1 Urease activity was determined using a colorimetric method, expressed as the mass (mg) of NH3-N generated per 1.0 g of soil after 1 day, and expressed in mg·g⁻¹. -1 ·d -1 Alkaline phosphatase activity was determined using the disodium phenyl phosphate colorimetric method. Phosphatase activity was expressed as milligrams of P₂O₅ in 1.0 g of soil after 1 day, and expressed in mg·g⁻¹. -1 ·d -1 The catalase activity was determined using the potassium permanganate titration method. Catalase activity was expressed as the number of milligrams of H₂O₂ consumed per minute per 1.0 g of soil, expressed in mg·g⁻¹. -1 ·min -1 express.

[0061] 1.9 Data Processing

[0062] Microsoft Excel 2010 was used for plotting and creating tables. SPSS Statistics 21 statistical software was used for data analysis. ANOVA was used for one-way ANOVA, and Duncan's method was used for multiple comparisons.

[0063] 2 Results and Analysis

[0064] 2.1 Pot experiment to test the effect of inoculant on wheat root rot

[0065] This study investigated the control effects of Z-14, HB-02, and their compound inoculants on wheat root rot. The results of the pot experiment are shown in the table below. The compound inoculant group ZHB showed higher root length, root weight, and aboveground weight than the control group CK0, but slightly lower aboveground height. All indicators were not significantly different from CK0, but significantly higher than the pathogen control group CK1. The Z14 treatment group showed lower root length, root weight, and aboveground height than the CK0 treatment group, but the differences were not significant. The aboveground weight was significantly lower than the CK0 treatment group. The HB02 treatment group showed significantly lower root length, aboveground height, and aboveground weight than the CK0 treatment group, and the root weight was also lower than the CK0 treatment group, but the differences were not significant. The control effect of the ZHB treatment group on wheat root rot reached 81.8%, significantly different from the 50.1% of the HB02 treatment group, but not significantly different from the 63.0% of the Z14 treatment group. In conclusion, the application of compound microbial agents can effectively prevent and control wheat root rot, and its effect is better than that of Z-14 and HB-02 microbial agents applied alone.

[0066] The effect of Bacillus preparations on the prevention and control of wheat root rot

[0067]

[0068] 2.2 Growth-promoting effect of Bacillus preparations on wheat

[0069] During the milk stage of wheat, the growth-promoting effects of Bacillus preparations on wheat were analyzed by investigating indicators such as plant height, number of spikes, number of grains per spike, thousand-grain weight, and yield. Compared with the blank control group CK0, the above indicators of wheat in the Z14, HB02, and ZHB treatment groups were all increased. There were no significant differences in any indicators between the Z14 treatment group and CK0. Compared with CK0, the HB02 treatment group showed a significant difference in yield, while the other indicators showed no significant differences. Compared with CK0, ZHB showed significant differences in plant height and yield, while the other indicators showed no significant differences. The ZHB treatment group was higher than the Z14 and HB02 treatment groups in all tested indicators, but the differences in each indicator were not significant. Compared with CK0, the Z14, HB02, and ZHB treatment groups increased yield by 7.51%, 14.69%, and 18.19%, respectively (see table below). Inoculation with Z-14 and HB-02 inoculants significantly increased wheat yield and had a significant growth-promoting effect, but the combined inoculant showed the best effect.

[0070] Detection of the growth-promoting effect of Bacillus preparations on wheat during the milk stage

[0071]

[0072]

[0073] 2.3 The effect of compound Bacillus preparation on the prevention and control of wheat root rot

[0074] This study investigated the control efficacy of different ratios of Z-14 and HB-02 compound microbial agents against wheat root rot. The results of the pot experiment are shown in the table below. The TR2 and TR3 groups showed higher root length, root weight, aboveground height, and aboveground weight than the CK0 group, but the differences were not significant. The TR2 group showed higher root weight than the TR3 group, but the difference was also not significant. The TR1 group showed significantly lower root weight than the CK0 group, while the other indicators showed no significant differences. The TR4 group showed significantly lower root weight than the CK0 group in all indicators, and the TR5 group showed significantly lower root weight than the CK0 group in all indicators, but the differences were not significant. Among the tested indicators such as root length, root weight, aboveground height, and aboveground weight, all treatment groups with the added compound microbial agents showed significantly higher root weight than the CK1 group, indicating that field Bacillus preparations can significantly inhibit the occurrence of wheat root rot. TR2 showed the best control effect against wheat root rot, with a control efficacy of 82.7%, followed by the TR3 group with a control efficacy of 79.1%, which is consistent with the results of the above four indicators. TR1 showed the lowest control efficacy at 45.9%, followed by TR4 at 53.6%. In conclusion, among the various compound microbial agents with different addition ratios, TR2 demonstrated the best control effect against wheat root rot and was able to effectively prevent its occurrence.

[0075] The effect of compound Bacillus preparations on the prevention and control of wheat root rot

[0076]

[0077]

[0078] 2.4 Growth-promoting effect of compound Bacillus preparation on wheat

[0079] During the milk stage of wheat, the growth-promoting effect of compound Bacillus preparations on wheat was analyzed by investigating indicators such as plant height, number of spikes, number of grains per spike, thousand-grain weight, and yield (see table below). Compared with the blank control group CK0, the plant height of the TR2, TR3, and TR5 treatment groups was significantly increased, and the plant height of the TR1 and TR4 treatment groups was also increased, but the differences were not significant. The number of spikes in the TR2 and TR4 treatment groups was significantly higher than that in the control group, and the number of spikes in the other treatment groups was also higher than that in the control group, but the differences were not significant. The number of grains per spike and thousand-grain weight of each treatment group with added inoculant were higher than those in the CK0 group, but the differences were not significant. The yield of each treatment group with added inoculant was significantly higher than that in the CK0 group. The TR2 treatment group had the highest yield, with a yield increase of 25.99% compared to the control group, followed by TR3, with a yield increase of 22.43% compared to the control group, and the lowest yield was in the TR4 group, with a yield increase of 13.77% compared to the control group. In conclusion, the TR2 treatment group had the best yield-increasing effect and a significant growth-promoting effect.

[0080] Test on the growth-promoting effect of compound Bacillus preparations on wheat during the milk stage

[0081]

[0082]

[0083] 2.5 Effects of compound Bacillus preparations on the activity of bioenzymes in wheat rhizosphere soil

[0084] The effects of compound Bacillus preparations on wheat growth promotion were investigated through field trials. At the wheat jointing stage, rhizosphere soil samples from different treatment groups were collected to measure the activities of sucrase, urease, alkaline phosphatase, and catalase. The results are shown in the table below. Compared with the blank control group CK0, sucrase activity increased in all treatment groups with the compound Bacillus preparation. The increase was not significant in the TR5 treatment group, but significant in the others. The TR2 group showed the largest increase in sucrase activity, reaching 36.64%, followed by the TR4 group at 32.24%. Urease activity increased to varying degrees in all treatment groups with the compound Bacillus preparation. The increase was not significant in the TR3 group, but significant in the others. The TR2 group showed the largest increase in urease activity, reaching 34.69%. Alkaline phosphatase activity increased in all treatment groups with the compound Bacillus preparation. The increases were not significant in the TR1 and TR5 groups, but significant in the others. The TR2 group showed the highest enzyme activity, increasing by 36.51%. All treatment groups treated with the compound microbial agent showed increased catalase activity, with significant increases in TR2 and TR4 groups, while the increases in the other groups were not significant. TR4 showed the largest increase (54.14%), followed by TR2 (43.95%). In summary, TR2 group showed the most significant increases in sucrase, urease, and alkaline phosphatase activities, with catalase activity second only to TR4 group, indicating that TR2 group had the most significant impact on enzyme activity in wheat rhizosphere soil.

[0085] Effects of compound Bacillus preparations on the activity of biological enzymes in wheat rhizosphere soil

[0086]

[0087] 3. Conclusion

[0088] The application of compound microbial agents effectively prevented wheat root rot, showing better results than the individual application of Z-14 and HB-02 agents. Both Z-14 and HB-02 inoculation significantly increased wheat yield and promoted growth, but the compound microbial agent showed the best effect. Among different proportions of compound microbial agents, the TR2 group showed the best control effect against wheat root rot, achieving a control rate of 82.7%, effectively preventing the occurrence of wheat root rot. The TR2 treatment group showed the best yield increase, increasing by 25.99% compared to the control group. The TR2 group showed the most significant increases in sucrase, urease, and alkaline phosphatase activities, with catalase activity second only to the TR4 group, indicating that the TR2 group had the most significant impact on wheat rhizosphere soil enzyme activity.

[0089] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A Bacillus species, characterized in that, It is a complex type of Bacillus, which includes: Bacillus subtilis ( Bacillus subtilis The accession number is CGMCC No. 20821; the depository center is the China General Microbiological Culture Collection Center; the deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is September 25, 2020. Gel-like Bacillus ( Paenibacillus mucilaginosus The accession number is CGMCC No. 24636; the depository center is the China General Microbiological Culture Collection Center; the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; and the deposit date is April 6, 2022.

2. A method for preparing a Bacillus-type compound microbial agent, characterized in that, Includes the following steps: S1: Strain selection and culture; S11: Bacillus subtilis Z-14, with strong growth vigor and resistance to stress and heat, was screened out (CGMCC No. 20821); and Bacillus mucilaginosus HB-02, with CGMCC No. 24636. The selected strains were streaked on NA medium and incubated overnight at an incubator. Activated single colonies were picked and transferred to NB medium and cultured overnight with shaking to prepare bacterial suspensions. S12: Bacterial activation, using NA medium for bacterial activation, and incubating overnight at a constant temperature; S13: Cell collection and preparation: Activated single colonies were inoculated into NB medium and cultured overnight with shaking. The culture was then inoculated into fresh NB medium at a 10% inoculation rate and cultured with shaking for 48 hours. The fermentation broth was centrifuged, the cells were recovered, and diluted with sterile water. S2: Preparation of compound microbial agents; S21: Determine the mixing ratio, conduct tests, and determine the optimal mixing ratio of Z-14 and HB-02 bacterial agents based on the test results; S22: Prepare the bacterial suspension by adjusting the viable bacteria content of the Z-14 and HB-02 bacterial agents, which are mixed in proportion, to 1.0 × 10⁻⁶. 10 A compound bacterial suspension was prepared by adding cfu / mL.

3. The method for preparing a Bacillus-type composite microbial agent according to claim 2, characterized in that, In step S11, the viable bacteria content of the bacterial suspension is controlled to be 1×10⁻⁶. 8 cfu / mL or 1×10 10 cfu / mL.

4. The method for preparing a Bacillus-type composite microbial agent according to claim 2, characterized in that, The specific components of the NA culture medium are: 5g beef extract, 10g peptone, 5g NaCl, 15-20g agar, and 1000mL water; The pH of the NA medium should be controlled between 7.0 and 7.2; The specific components of the NB culture medium are: 5g beef extract, 10g peptone, 5g NaCl, and 1000mL water; The pH of NB medium should be controlled between 7.0 and 7.

2.

5. The method for preparing a Bacillus-type composite microbial agent according to claim 2, characterized in that, In step S13, the centrifugation speed is controlled at 8000 rpm and the centrifugation time is controlled at 10 minutes.

6. The method for preparing a Bacillus-type composite microbial agent according to claim 2, characterized in that, In step S13, sterile water is used to dilute the solution to a viable bacteria content of 1×10⁻⁶. 8 cfu / mL or 1×10 10 cfu / mL.

7. The method for preparing a Bacillus-type composite microbial agent according to claim 6, characterized in that, In step S21, the specific methods for conducting the test and detection include: Pot experiment: A pot experiment was conducted by setting up a blank control group, a pathogen group, and different treatment groups to test the control effect and growth-promoting effect of each treatment on wheat root rot. Field trials: Field trials were conducted under actual farmland conditions to determine the effects of compound microbial agents on wheat yield, plant height, number of ears, number of grains per ear, and soil enzyme activity. Effect analysis and ratio optimization: Combining the results of pot and field trials, the effects of different proportions of compound microbial agents were analyzed, and the optimal mixing ratio was finally determined.

8. The method for preparing a Bacillus-type composite microbial agent according to claim 2, characterized in that, In S21, the optimal mixing ratio is Z-14 and HB-02 bacterial agents mixed at a ratio of 2:

1.

9. The method for preparing a Bacillus-type composite microbial agent according to claim 7, characterized in that, In the pathogen group, the pathogen culture medium used is PDA medium, which is specifically composed of: 200g potato, 20g glucose, 15-20g agar, and 1000mL water. Peel the potato, cut it into small pieces, boil it in water for 0.5h, filter it with double-layer gauze, take the filtrate, add sugar, and add water to make up to 1000mL. Melt the sterilized PDA medium and pour it into plates. Add streptomycin sulfate to make the final concentration 40 μg / mL. Inoculate the pathogenic bacteria slices in the center of the PDA plates and incubate at 26°C until the mycelium has fully grown on the plates.

10. A Bacillus-type compound microbial agent, characterized in that, It is prepared by the preparation method according to any one of claims 2-9.

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